Investigations in this proposal are focused on identifying the molecular basis for the ligand dependent and independent actions of the VDR, using the skin as a model system. Like humans with VDR mutations, VDR null mice develop alopecia. We have demonstrated that the ligand-independent actions of the VDR are required for hair follicle keratinocyte stem cell (KSC) function, normal hair cycling and canonical Wnt (cWnt) signaling in keratinocytes. Interfering with cWnt signaling also impairs KSC function. Studies examining the interactions of the VDR with effectors of canonical Wnt signaling demonstrate that the VDR interacts with Lef1 via its DNA binding domain and that the VDR interacting domain of Lef1 is independent of its [unreadable]-catenin binding domain. We will examine the functional consequences of impairing VDR-Lef1 interactions in keratinocytes and determine if the interaction between these two proteins is direct, or involves additional factors. While keratinocyte-specific expression of a VDR transgene rescues the hair cycle defect in VDR null mice, a constitutively active [unreadable]-catenin transgene does not, placing the VDR at the level of, or downstream from activation of cWnt signaling. Initiation of hair follicle growth (anagen) by cWnt signaling induces Shh and Gli1 mRNA in WT mice but not in VDR-/- mice. Furthermore, the expression of Shh, Gli1 and the classic cWnt target gene c-myc, is impaired in the skin of VDR-/- mice. Although Shh is not expressed in primary keratinocytes, Gli1 and c-myc mRNA levels are reduced in VDR-/- keratinocytes and restored by VDR transfection. ChIP analyses demonstrate that the VDR interacts with Gli1 regulatory sequences. Studies in keratinocytes from WT, VDR-/- and Lef1-/- mice will address the hypothesis that the unliganded VDR and Lef1 interact with regulatory regions of Gli1 in the context of intact chromatin and are required for basal and Wnt3a-induced expression of this gene. We will also examine if activation of the HH pathway induces anagen in VDR-/- mice. The VDR-/- mice also exhibit a defect in wound healing. Preliminary studies point to defects in macrophage recruitment and vascular invasion, which are also observed in vitamin D deficient WT mice, demonstrating that these reflect impaired ligand-dependent actions of the VDR. We will identify the cellular and molecular basis for the abnormalities observed and determine which actions of the VDR required for wound repair are ligand dependent. The goal of these studies is not to study skin biology per se, but rather to characterize the cellular and molecular basis for in vivo findings to identify novel actions of the liganded and unliganded VDR. PUBLIC HEALTH RELEVANCE: Impaired Vitamin D action, associated with Vitamin D deficiency is an increasingly recognized public health problem. While several consequences of impaired Vitamin D action are well understood, many are not. The studies in this proposal will employ a mouse model of the human disorder, Hereditary Vitamin D Resistant Rickets, to identify the molecular basis for abnormalities that result from impaired ligand-independent actions of the vitamin D receptor in the hair follicle and impaired ligand-dependent actions in cutaneous wound healing. These studies are expected to reveal novel functions for this receptor in keratinocytes stem cells as well as in the inflammatory and angiogenic stages of cutaneous wound repair, thus have implications for other disorders associated with impaired wound healing, including diabetes mellitus.